Cannabinoids, initially thought to act through non-specific interactions related to cell membrane fluidity, were shown to activate specific cannabinoid receptors. Two types of these receptors exist, CB1, most expressed in the brain, and CB2 mostly expressed in the periphery. Cannabinoids have been shown to activate other receptors, for example, TRPV1, PPAR, and GPR55. Different endogenous compounds bind to cannabinoid receptors, among them, anandamide (AEA), and 2-arachidonyl-glycerol (2AG). These compounds are not stored in vesicles, like other transmitters, but are released on demand by cleavage of membrane precursor components. The recent availability of natural and synthetic ligands, and drugs that block the metabolism or the uptake of endocannabinoids, has facilitated the study of the role of cannabinoids in physiological functions, for example in synaptic plasticity. Dysfunctions of the endocannabinoid system could also result in neurological disorders, including drug addiction. Collaborating with Drs. Goldberg and Solinas we have shown that the endocannabinoids AEA (and its metabolically stable analogue methAEA) and 2AG share with drugs abused by humans the ability to acutely stimulate DA levels in the rat NAC shell, suggesting endocannabinoids may possess reinforcing effects. Indeed, AEA promotes and maintains intravenous self-administration behavior in monkeys. These effects are blunted by pretreatment with a selective CB1 antagonist, indicating a direct CB1 receptor involvement in elevation of DA and reinforcing effects. Compounds known to increase brain endocannabinoid levels may also elicit DA release. URB597 inhibits the enzyme Fatty Acid Amide Hydrolase (FAAH) that metabolizes AEA. This drug alone (at doses that fully block FAAH) did not produce DA stimulation, suggesting that, in animals at rest, levels of AEA are not high enough to activate CB1 receptors, even after FAAH blockade. Alternatively, FAAH might not be sufficiently expressed in areas related to DA transmission. However, URB597 enhanced the effects of exogenous AEA on DA levels, supporting the hypothesis of low levels of AEA in animals at rest, and suggesting that URB597 can block AEA metabolism resulting in increased levels of AEA in areas related to DA transmission. URB597 alone does not elevate DA levels, and its degree of abuse liability appears low, in agreement with negative findings of self-administration in monkeys, and place preference in rats. AM404, another endocannabinoid enhancer, produces cannabinoid-like behaviors in rodents, but in our experiments it did not alter DA levels, and surprisingly, did not enhance the effect of AEA on NAC shell DA. In agreement, in rats trained to discriminate THC from saline (a behavior selectively mediated by CB1 receptors), AEA alone did not produce full THC-like effects (likely due to its rapid metabolism), while after pretreatments with URB597, but not with AM404, AEA produced full THC-like effects. DA D2 receptor activation stimulates AEA levels, thus, drugs that increase DA levels in the brain, like abused drugs, can activate DA D2 receptors, and in turn, increase AEA levels. We have tested the effects of abused drugs in producing CB1-receptor mediated generalization in THC-discrimination tests. Cocaine, and amphetamine injected alone did not produce effects significantly different from vehicle, but potentiated the THC-like effects of THC. Nicotine and the D2/3 DA receptor agonist quinpirole alone did not generalize to the THC cue, but both drugs did so in animals pretreated with the inhibitor of FAAH that metabolizes AEA. Nicotine and quinpirole also potentiated the effects of THC. We have suggested that AEA is released by these drugs in specific brain areas by a D2 receptor mediated mechanism. So, administered alone these drugs do not stimulate AEA levels sufficiently to provide CB1-mediated THC-like effects, but potentiate ineffective small doses of THC. However, when the same drugs are administered in combination with URB-597, AEA levels are magnified by blockade of its metabolism, and its concentration could thus activate CB1 receptors producing THC discriminative effects. Recently it has also been shown that brain actions of anandamide and blockers of anandamide metabolism (such as URB-597) might be mediated not only by the endocannabinoid system, but also by PPAR- receptors. It has also been demonstrated that blockade of anandamide metabolism through fatty acid amide hydrolase enzymes by URB-597 might lead to increased levels of oleoylamide (OEA) or palmytoilamide (PEA), as well as anandamide. While anadamide has both CB1 and PPAR- receptor activities, OEA and PEA are selective ligands for PPAR receptors, with negligible activity at CB1 receptors. In a recent publication in collaboration with Drs. Paola Mascia and Steven Goldberg it has been shown that drugs acting specifically at brain PPAR- receptors can block the addictive actions of nicotine in rats and monkeys, using self-administration and reinstatement behavioral models of nicotine dependence. Psychostimulant sensitization might play a role in the path to abuse and addiction, and even a single exposure to psychostimulants could produce long-term sensitization by increasing strength of excitatory synapses in midbrain dopaminergic regions. Sensitization can be viewed as a type of synaptic plasticity, which is also related to alterations in the cannabinoid system. We hypothesized that development of psychostimulant sensitization involves changes in endocannabinoid levels and their activity at CB1 receptors. We started this project studying cocaine sensitization in mice (measured as increased stimulation of behavioral activities, for example distance traveled by mice, before and after sensitizing doses of cocaine). We are testing the hypothesis that the development of cocaine sensitization requires release of endocannabinoids, and can be reversed by CB1 receptor blockade. Based on our original hypothesis, low doses of cocaine that do not induce behavioral sensitization might become effective when animals are pretreated with enhancers of endocannabinoid levels. DA transmission, believed to mediate behavioral and reinforcing effects of cocaine, will also be measured before and after cocaine sensitization. Preliminary results from this study have confirmed that a single exposure to 20 mg/kg of cocaine induces behavioral sensitization in mice. Rimonabant, a CB1 antagonist, injected before the sensitizing dose of cocaine, reduced the sensitization produced by cocaine. In addition, cocaine-induced sensitization is paralleled by a sensitization of the DA response in the nucleus accumbens core, whereas no change were found in the NAC shell. Preliminary studies suggest that blockade of endocannabinoid metabolism (obtained by pretreatment with URB-597 in mice) enhances the extracellular levels of endocannabinoids released by cocaine, and this enhancement could be related to the induction of behavioral sensitization by doses of cocaine otherwise not effective. We are also studying the role of CB1 receptors on the reinforcing effects of food. This study uses genetically modified CB1-receptor KO mice and their wild-type littermates. Mice are trained to emit operant responses to get a food reward. As the number of required responses is increased, food consumption decreases. This demand function is derived from behavioral economic theory, and has been validated as a behavioral index of reinforcing efficacy. Studies are underway to evaluate the role of CB1 receptors in the value of specific reinforcers using these genetically engineered mice.